One of the most commonly observed effects of opioids is their ability to inhibit the release of neurotransmitters in both the peripheral and central nervous systems ("presynaptic inhibition"). We have explored the possible mechanisms by which opioids might produce these effects. In particular, we have examined the hypothesis that activation of opioid receptors may inhibit the influx of Ca into neurons -possibly as a result of the direct inhibition of those Ca channels that are coupled to neurotransmitter release. We demonstrated that neurons possess a variety of Ca channel types and that these have different degrees of influence on the release of neurotransmitters. Activation of opioid receptors produces inhibition of the same types of Ca channels that are coupled to neurotransmitter release. In the current proposal, we shall continue to study this phenomenon by carrying out the following types of studies (I). We shall examine the mechanisms by which opioid receptor activation produces inhibition of different types of Ca channels in acutely isolated neurons of the nucleus tractus solitarius of the rat. This is an area of the brain in which opioids are thought to produce many of their effects on autonomic function (2). We shall investigate the coupling between opioid receptors and Ca channels by constructing cell lines that possess opioid receptors and Ca channels. For example, we have constructed cell lines that contain N type Ca channels and the kappa-opioid receptor (3). We shall investigate the coupling between kappa-opioid receptors and the inwardly rectifying K channel GIRK-1 by constructing cell lines that contain these proteins (4). We shall investigate the ability of opioid receptors to produce presynaptic inhibition and modulation of ion channels when they are expressed in normally opioid insensitive neurons. Opioid receptors will be introduced into these cells by using replication defective adenovirus. These studies should greatly increase our understanding of the way opioids can regulate the activity of neurons.